Abstract Nonsyndromic orofacial clefts (OFCs) are the most common craniofacial birth defects in humans, affecting approximately 1 in 700 individuals worldwide. Genome-wide linkage and association studies have identified several risk alleles for OFCs; however, they account for a minority of their estimated heritability. Therefore, while the ongoing genetic and expression studies may lead to important advances in understanding the biological basis underlying OFCs, a fuller picture will only emerge as the interaction of susceptibility variants with other factors, such as epigenetic changes, are established. Epigenetic modification is a likely mechanism through which environmental factors and genetic variation may alter gene expression. DNAm is a covalent addition of a methyl (CH3) group to the nucleotide cytosine, which can lead to changes in transcriptional activity of the targeted gene. We hypothesize that changes in methylation and the resulting differential gene expression is an epigenetic risk factor for OFCs, and that DNAm mediates genetic influences in OFCs risk. Our main goals are to characterize genome-wide DNAm variation that can impact the risk for OFCs; and explore the genetic-epigenetic interactions (meQTLs) involved in the etiology of OFC. To do so, this project proposes a powerful strategy using the largest sample set (to date) of monozygotic twins and sibling pairs discordant for nonsyndromic OFCs used to study epigenetic risk factors. This is a key step and an innovative approach that will allow uncovering epigenetics risk factors that are invisible to conventional GWAS and DNA sequencing methods. The central hypothesis of this proposal will be tested with the following specific aims: (1) To determine genome-wide DNA methylation patterns in MZ twins discordant for OFCs; (2) To explore the genetic-epigenetic interactions (meQTLs) involved in the etiology of OFCs. Within aim 2 we will attempt to replicate the genetic-epigenetic interactions identified in the preliminary analysis (obtained since the first submission of this proposal), and top hits resulting from Aim 1, using an independent cohort of cleft discordant sibling pairs. The outstanding team of mentors (Dr. Jeff Murray, Dr. Robert Philibert, Dr. Mary Marazita, and Dr. Moreno-Uribe), consultants (Dr. Xie, and Dr. Drake), collaborator (Dr. Lie), and advisors (Dr. Amendt, Dr. Wehby and Dr. Butali) assembled in this proposal covers all areas of the much needed additional training necessary to accomplish the proposed research and training aims. They will provide guidance and facilitate the growth of the PI during the transition to independence. Although very well trained initially, the five years hiatus in the PI's career justify the need for additional training in the rapidly moving fields of epigenetics and big data applications. The proposed training and research aims are tailored to build upon the PI's previous experience and to provide the additional training in epigenetics and analytical methods necessary to propel the PI's development as an independent researcher able to integrate epigenetic and genetic data to study complex craniofacial birth defects.